The alkyl hydroperoxide reductase (AhpR) enzyme system of Salmonella typhimurium serves to protect these organisms from the toxic and mutagenic effects of oxidative stress. The long-term objective of these proposed studies is to elucidate the catalytic mechanisms for each of the two proteins of the AhpR system, AhpC and AhpF, in order to gain a better understanding of the physiological role these proteins play in protecting cellular macromolecules against reactive oxygen species. A broader objective is to define how widely this enzymatic activity is distributed among aerobic and anaerobic organisms. Ubiquity of this system has been suggested by the identification of a number of AhpR homologues from a wide variety of sources. The primary hypothesis to be tested is whether or not redox-active cysteine residues are involved in the heme-independent catalysis of peroxide reduction, as might be expected given mechanisms of two other known non-heme peroxide reductases, NADH peroxidase and glutathione peroxidase. Specific aims include l) demonstration of the specific catalytic roles of each AhpR protein and identification of catalytically important amino acids, including cysteine residues, within each protein, 2) determination of whether or not the 207 amino acids at the N-terminus of AhpF, which have no counterpart in the otherwise homologous thioredoxin reductase protein, are required for catalysis or serve any other discernible function, 3) structural characterization of each protein, including X-ray crystallographic studies if high-quality crystals can be generated, and 4) analysis of the ability of other AhpC homologues to support peroxide reduction. Efforts to address these specific aims will include thermodynamic and kinetic studies of the enzymatic activities of each protein in conjunction with chemical modification and site-directed and random mutagenesis studies. The AhpR system is of considerable interest in terms of its chemistry as another possible example of the participation of an unusual oxidized form of cysteine, cysteine sulfenic acid (R-SOH), in catalysis. One aspect of the physiological importance of AhpR may be in helping pathogenic Salmonella, and possibly other human pathogens as well (including Entamoeba histolytica, Helicobacter pylori and Mycobacterium avium), to escape killing by toxic oxygen species produced by the host's phagocytes. The identification of this enzyme system in a wide variety of organisms, as has been suggested by the wide distribution of Ahp homologues, may lead to a more generalized significance for AhpR as an antioxidant enzyme system. Antioxidant systems are critical in higher organisms in countering such oxidation-linked processes as carcinogenesis, inflammation and age-related diseases.